mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Metagenomic analysis in combination with high through put sequencing: An analysis of Resistance and Gene transfer in antibiotic contaminated rivers
In this article the authors identify resistance genes and elements for horizontal gene transfer such as integrons, transposons and plasmids in the microbiome of river sediments at both upstream and down stream sites of pharmaceutical manufacturing plants in India. The authors make use of shotgun metagenomics and high throughput sequencing techniques to identify enzymes and genes involved in haorizontal gene transfer such as Class 1 integrase and ISCR transposase class 2 1 Introduction The over use of antibiotics in developing countries for the purposes of human and veterinary medicine promotes the development and spread of multiresistant bacteria. Even small levels of anitbiotic release into the environment results in increased selection pressure 2. High levels of antibiotic resistance gene were found in variety of environment particularly those exposed to anitbiotics, fertilizers and in river waters contaminated with runoff water from farms.A recent study showed that oxytetracycline was detected upto 20mg/L in the treated effluent from a pharmaceutical plant in China and to about 31 mg/L fluoroquinoline class of antibiotics such as cirprofloxacin in the downstream waters collected in India Fig 1. Antibiotics are also naturally produced in the environment by fungi actiniomycetes bacteria have evolved different resistance genes, most of which are mobile and able to cross and spread to different species of bacteira and confer resistance to them. Recent studies have coined the term RESISTOME demonstrating the ever increasing and diverse pool of resistance genes in the bacterial communities1. And thus high levels of anitbiotics in the enviroment ultimately leads to rapid and global transmission of resistant pathogens. And hence the Authors feel that the consequences of high environmental release of antibiotics should be addressed. Methods. #Samples were collected from up and down stream for waste water treatment plant in Hyderabad (India) and Skovde (Sweden) in a gradient and were pooled. #Sequencing was done using a Roche genome sequencer FLX Titanium and the metagenomic data was depositied in NCBI Sequence Read Archive ( Accession Number SRP002078). #Chemical analysis of antibiotics were performed by mass specterometry. #All the sequencing reads were compared against the non-redundant protein BLAST (NCBI BLAST) and taxonimic affiliation was extracted from the NCBI Taxonomy database. #Functional annotation was done by comparing the reads against the Clusters of Orthologus Genes and TIGRFAMS database using RPSBLAST. #A read was annotated as a resistance gene according to its best BLAST hit. #Metagenomics were searched for qnr-like resistance genes using a position specific scoring matrix (PSSM) from five classes of known qnr-,qnrS1 using ClustalW 2.0. Results High levels of ciprofloxacin was detected in the sediments collected down stream and measured upto 914microgram/gram organic matter in samples collected from India Fig1. The sediment DNA was analyzed by multiplexed massively parallel pyrosequencing and the results suggested that of the 441,523 reads, 85.5% of the annotated sequences were of bacterial origin. Functional analysis showed significant differences in metagenomes and that the downstream bacterial communities contained high abundance of antibiotic resistance genes such as COG0294, genes associated with replication and mobilization of DNA such as COG3668 etc. A detailed charaterization of the antibiotic resistance showed significant differences in abundance for genes that confer resistance to sulfonamides, fluoroquinolones and aminoglycosides (fig2) . The most abundant gene identified was sul2 gene which confers sulfonamide resistance variant ofdihydropteroate synthase . Other resistance genes including strA and strB were found be 22 and 54 times higher in downstream sediments in the Indian river samples. strA and strB genes code for aminoglycoside ohosphotransferase which inactivates streptpmycin by phosphorylation. The relative abundance of ClassI integrases was found to significantly higher downstream compared to upstream from the indian water treatment plant Fig3 . Integrons are known to capture and disseminate short circular pieces of DNA which may harbour resistance genes. Their study also revealed that the water samples from the downstream contained higher levels of transposase associated with insertion sequence common regions (ISCR) which mobilize larger segments of DNA.Two plasmids RSF 1010 and pMTSm3 which carry sul2 and strA/B resistance and class 2 ISCR transposase was also found to higher in downstream waters. Over all this study shows that multiple resistance genes were transfered in a high antibiotic environment and the role of unintended antibiotic release which could lead to potential recrutiment pools for pathogens to gain antibiotic resistance. 1 Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements Erik Kristiansson, Jerker Fick, Anders Janzon, Roman Grabic, Carolin Rutgersson, Birgitta Weijdegård, Hanna Söderström, D. G. Joakim Larsson mail Published: Feb 16, 2011DOI: 10.1371/journal.pone.0017038 2 Antibiotic resistance genes in water environment.